Peptide or collection of peptides derived from amyloid precursor protein

ABSTRACT

The present invention relates to a peptide or collection of peptides derived from amyloid precursor protein (APP). The present invention also relates to uses of such peptide or collection of peptides, in particular as a diagnostic marker and/or as a drug target.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. Ser. No.15/559,613, filed Sep. 19, 2017; which is a National Stage Applicationof International Application Number PCT/EP2016/055081, filed Mar. 10,2016; which claims priority to European Patent Application No.15159877.8, filed Mar. 19, 2015; all of which are incorporated herein byreference in their entirety.

The Sequence Listing for this application is labeled“SeqList-14Sep17-ST25.txt”, which was created on Sep. 14, 2017, and is15 KB. The entire content is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates to a peptide or collection of peptidesderived from amyloid precursor protein (APP). The present invention alsorelates to uses of such peptide or collection of peptides, in particularas a diagnostic marker and/or as a drug target.

BACKGROUND OF THE INVENTION

Intensive efforts are on the way to develop therapeutic strategiestargeting Aβ, the major component of the Alzheimer's disease signifyingamyloid plaques¹. AP is generated from APP by proteolytic processing.First, β-secretase, identified as BACE1, cleaves at the Met-Asp bond ofthe AP domain and generates CTF-β, which is further processed to Aβ byintramembrane cleavage by γ-secretase⁶. Liberated Aβ forms solublesynaptotoxic oligomers, which are believed to be the major culprit ofthe disease¹. Thus targeting the production of synaptotoxic Aβ speciesby β-secretase inhibition is a promising therapeutic strategy andclinical studies with high affinity BACE1 inhibitors are currentlyevaluated^(7,8). Decreasing BACE1 activity leads to an increase ofnon-amyloidogcnic processing via ADAM10⁹. Similarly, enhancingα-secretase activity reduced Aβ production and plaque formation¹⁰.However, stable isotope labeling kinetics upon in vivo inhibition ofBACE1 in monkeys revealed an 83% decrease of sAPP-β but only a 35%increase of sAPP-α³. Thus, the fate of almost 50% of the initiallylabeled APP remains unclear. In addition to the two major andwell-studied proteolytic processing pathways, APP is also shed in minorprocessing pathways utilizing different proteases¹¹. Furthermore, 17-35kDa N-APP fragments are generated during early development and upontrophic-factor deprivation¹²⁻¹⁴. However, such alternative APPmetabolites were not observed to accumulate upon BACE1 inhibition.

BRIEF SUMMARY OF THE INVENTION

Hence, there is a need in the art to identify new pathways that areinvolved in the genesis of Alzheimer's disease. More specifically, thereis a need in the art to identify molecular species which may eitherserve as a diagnostic marker or a drug target, more specifically, as adiagnostic marker or drug target for Alzheimer's disease.

BRIEF DESCRIPTION OF THE FIGURES

Furthermore, reference is made to the figures, wherein

FIGS. 1A-1H present data on the proteolytic processing pathway of APP inaccordance with the present invention;

FIGS. 2A-2C provide data on the physiological role ofmatrix-metalloproteinase MT5-MMP;

FIGS. 3A-3D show data with respect to the inhibition of β-secretase(BACE1);

FIGS. 4A-4E show data on the effects of peptide in accordance with thepresent invention, Aη-α and Aη-β on hippocampal long term potentiation(LTP);

FIG. 5 shows a table on antibodies that were used in the experimentalwork leading to the present invention;

FIGS. 6A-6F show immunohistochemical data on dystrophic neurites inbrains affected by Alzheimer's disease, using antibodies againstAη-epitope(s);

FIG. 7 shows the effects of Aβ_(S26V)-dimers on long term potentiation(LTP).

FIGS. 8A-8B show the effects of overexpression of MT1-MMP and MT5-MMP inN2a cells.

DETAILED DESCRIPTION

In a first aspect, the present invention relates to a peptide orcollection of peptides derived from amyloid precursor protein (APP) bythe action of matrix-metalloproteinase MT1-MMP or MT5-MMP, and by the(further) action of at least one enzyme selected from α-secretase(ADAM10) and β-secretase (β-site APP cleaving enzyme 1; BACE1).

In one embodiment, the peptide or collection of peptides according tothe present invention is derived from amyloid precursor protein (APP) bythe action of matrix-metalloproteinase MT1-MMP or MT5-MMP, and bothα-secretase (ADAM10) and 3-secretase (β-site APP cleaving enzyme 1;BACE1).

In one embodiment, the peptide or collection of peptides has one orseveral epitopes selected from MISEPRISYG (SEQ ID NO: 1), DALMPSLT (SEQID NO: 2), PWHSFGADSVP (SEQ ID NO: 3), SEVKM (SEQ ID NO: 4), andDAEFRHDSGYEVHHQK (SEQ ID NO: 5).

In one embodiment, the peptide or collection of peptides ischaracterized by recognition through an antibody which recognizes one orseveral of said aforementioned epitopes.

In one embodiment, the peptide or collection of peptides has a sequenceselected from MISEPRISYGNDALMPSLTETKTTVELLPVNGEFSLDDLQPWHSFGADSVPANTENEVEPVDARPAADRGLTTRPGSGLTNIKTEEISEVKM (SEQ ID NO: 6) and MISEPRISYGNDALMPSLTETKTTVELLPVNGEFSLDDLQPWHSFGADSVPANTENEVEPVDARPAADRGLTTRPGSGLTNIKTEEISEVKMDAEFRHDSGYEVHHQK (SEQ ID NO: 7).

In one embodiment, one peptide has a sequenceMISEPRISYGNDALMPSLTETKTTVELLPVNGEFSLDDLQPWHSFGADSVPANTENEVEPVDARPAADRGLTTRPGSGLTNIKTEEISEVKM (SEQ ID NO: 6) and another peptidehas a sequence MISEPRISYGNDALMPSLTETKTTVELLPVNGEFSLDDLQPWHSFGADSVPANTENEVEPVDARPAADRGLTTRPGSGLTNIKTEEISEVKMDAEFRHDSGY EVHHQK (SEQ IDNO: 7).

In a further aspect, the present invention relates to a composition ofpeptides comprising one or several peptides according to the presentinvention and at least one further peptide derived from amyloidprecursor protein (APP) by the action of matrix-metalloproteinaseMT1-MMP or MT5-MMP only.

In one embodiment, the at least one further peptide is membrane bound oris soluble in aqueous solution.

In one embodiment, the composition according to the present inventioncomprises both a membrane bound further peptide derived from amyloidprecursor protein (APP) by the action of matrix-metalloproteinaseMT1-MMP or MT5-MMP only and a peptide soluble in aqueous solution andderived from amyloid precursor protein (APP) by the action ofmatrix-metalloproteinase MT1-MMP or MT5-MMP only.

In one embodiment, said membrane bound further peptide has a sequencerepresented by SEQ ID NO: 8, and said peptide soluble in aqueoussolution has a sequence represented by SEQ ID NO. 9.

The invention also relates to the peptide or collection of peptides orthe composition of peptides according to the present invention, for useas a diagnostic marker.

In one embodiment, said use involves the detection of said peptide or ofsaid collection or composition of peptides in cerebrospinal fluid (CSF)or plasma.

In one embodiment, said diagnostic marker is a marker for Alzheimer'sdisease.

The present invention furthermore relates to the peptide or collectionof peptides or the composition of peptides according to the presentinvention, for use as a drug target, preferably as a drug target in thetreatment, prevention and/or alleviation of Alzheimer's disease.

In one embodiment, said use as a drug target involves the inhibition offormation or action of said peptide or said collection of peptides or ofsaid composition of peptides, or the inhibition of action or productionof matrix-metalloproteinase MT5-MMP and/or MT1-MMP.

As used herein, a “peptide derived from amyloid precursor protein (APP)by the action of one or several matrix-metalloproteinases” is meant torefer to a peptide that is the reaction product of the one or severalmatrix-metalloproteinases acting on the amyloid precursor protein andcleaving the same into fragment(s). The present inventors havesurprisingly identified a novel prominent proteolytic pathway which isinitiated by the action of matrix-metalloproteinase 5 and/ormatrix-metalloproteinase 1 or both. As used herein, the action of suchmatrix-metalloproteinase 5 and/or 1 is herein also sometimes referred toas “η-secretase”. The present inventors believe that this novelproteolytic pathway is initiated by such η-secretase and is followed bythe action of other secretases, most notably α-secretase (ADAM10) and/orβ-secretase (β-site APP cleaving enzyme 1; BACE1). The resultantpeptides are herein also sometimes referred to as “Aη-peptides” whichcomprise Aη-α and Aη-β (see also FIG. 1a and FIG. 8). These Aη-peptidesturn out to inhibit hippocampal long-term potentiation (LTP), similar tothe familiar Aβ-oligomers. Furthermore, the Aη-peptides accumulate upongenetic and pharmacological inhibition of β-secretase (BACE1). Thepeptides thus identified are useful as diagnostic markers and/or as drugtargets. In particular, they are useful as diagnostic markers forAlzheimer's disease and as drug targets in the treatment, preventionand/or alleviation of Alzheimer's disease.

The present invention also relates to the use of the peptide(s) orcollection of peptides according to the present invention for themanufacture of a medicament for the prevention, treatment and/oralleviation of Alzheimer's disease. In one embodiment, said use involvesthe administration of a compound to a patient, which compound inhibitsthe formation or action of one or several peptides according to thepresent invention. Preferably, said patient is a human patient.

The present invention also relates to a method of identifying a compounduseful for the prevention, treatment and/or alleviation of Alzheimer'sdisease, wherein said compound is identified based on its capability toinhibit the formation or action of one or several peptides according tothe present invention.

The present invention also relates to a method of detecting Alzheimer'sdisease in a patient, wherein said method involves detecting one orseveral peptides in accordance with the present invention, i. e.peptides, derived from amyloid precursor protein (APP) by the action ofmatrix-metalloproteinase MT1-MMP or MT5-MMP, and by the action of atleast one enzyme selected from α-secretase (ADAM10) and β-secretase(β-site APP cleaving enzyme 1; BACE1). In one embodiment, said detectionoccurs by detecting said peptide(s) in cerebrospinalfluid (CSF) and/orplasma. Preferably said patient is a human patient.

The present invention also relates to a method of treatment, preventionand/or alleviation of Alzheimer's disease, wherein said method involvesthe inhibition, in a patient, of the formation or the action of thepeptide(s) in accordance with the present invention, or it involves theinhibition of the action or production of matrix-metalloproteinaseMT5-MMP and/or MT1-MMP. In one embodiment, the inhibition of theformation or action of said peptide(s) is achieved by the administrationto a patient of a drug that inhibits the formation of said peptide(s).In another embodiment, said method of treatment involves theadministration to a patient of a drug that inhibits the action of saidpeptide(s). Inhibition of the formation of said peptide(s) may forexample occur by binding to APP and/or to the enzyme(s) cleaving saidAPP. The inhibition of the action of said peptide(s) may occur bybinding to said peptide(s) or to its (their) respective bindingpartner(s). Preferably, said patient is a human patient.

The specific sequences disclosed herein refer to and are derived fromthe specific splicing variant P05067-4 (as disclosed in the data baseUniProt, human isoform APP695 of amyloid beta A4 protein). It should beclear, however, that the peptide(s) according to the present inventionare not limited to the peptides derived from this specific splicingvariant, and a person skilled in the art will be able to identifycorresponding equivalent peptides from other splicing variants as well.It should also be noted that the sequences used herein are humansequences, despite the fact that some of the experiments performedherein were done with mice. However, the data obtained in FIGS. 3-4, 6-8were obtained with human sequences, and there is an extremely highhomology between the mouse and human sequences, lending further supportto the general applicability of the present inventors' findings.

More specifically,

FIG. 1 shows a novel proteolytic processing pathway of APP and the datain respect thereof

a) Schematic representation of the η-secretase pathway (left) andpreviously known amyloidogenic pathway (right). Antibodies used in thisstudy are indicated (see also FIG. 5). b) A novel 30 kDa N-terminallyelongated APP-CTF-η fragment is detected in membrane fractions obtainedfrom brains of adult (22 month) and postnatal day 10 (P10) mice usingthe rabbit monoclonal antibody Y188 directed against the C-terminus ofAPP. CTF-η is specifically found in young and old wild type (WT) micebut absent in APP knockout mice (APPKO). In addition to this novelfragment, Y188 is detecting CTF-β and CTF-α. Full-length APP (APP-FL)was detected with antibody 22C11. β-Actin served as a loading control.c) Aη was identified as several closely spaced peptides detected in thesoluble (DEA) fraction of adult age and P10 mice by antibody M3.2. Asimilar pattern is detected by antibody 9478D that is specificallyrecognizing an N-terminal part of the Aη peptide. AT signal is moreprominent in P10 mice compared to adult mice. Detection of sAPP-α andsAPP-β species is shown as additional control. APPKO brain were used ascontrols for antibody specificities. β-Actin served as a loadingcontrol. d) Aη and Aβ were readily detectable in 10 μl of human CSF byantibody 2D8 at similar intensities. The antibody 2E9 allowed theselective detection of Aη in the same samples. e) Soluble (DEA) extractsof APPPS-21 mouse brains contained Aη species detected by 2E9. Aη-β(swe)was selectively detected by antibody 192swe in addition to sAPP-β(swe).While 2D8 antibody detected robust levels of sAPP-α, only low levels ofAη-α could be detected in APPPS-21 brain lysates due to theoverexpression of APPswe transgene. f) An increase in CTF-η is observedin RIPA lysates of APPPS-21 mouse brains (long exposure) using antibodyY188 as compared to WT. Full-length APP (APP-FL) was detected withantibody 22C11. β-Actin served as a loading control. g)Immunohistological stainings of cortical sections of 6 months oldAPPPS-21 transgenic mice revealed 6E10 positive Aβ plaque cores(encircled) surrounded by dystrophic neurites positive for 2E9 (whitearrowheads, upper panel) and 9476M (white arrowheads, middle panel).Y188 (lower panel) staining co-localized with 2E9 positive signal(yellow arrowheads, lower panel). Nuclei were counterstained with DAPI.Calibration bar=10 μm. h) Accumulation of CTF-η fragment in dystrophicneurites of 14 months old APPPS-21 mice. LCM of APPPS-21 brain sectionsbearing Thioflavin-S positive Aβ plaque core (P) and the surrounding Aβplaque halo (H) was performed. As control (C) brain areas, devoid ofplaques, were used. While Aβ was readily detected by antibody 2D8 inlysates containing plaque enriched material and halo regions (fractionsP and H), CTF-η was selectively detected in the lysates prepared fromthe region enriched in dystrophic neurites (H), but not detected inplaque or control regions (P and C). As expected, CTF-β/α species arealso enriched in dystrophic neurites (H).

FIG. 2 shows that MT5-MMP has physiological TI-secretase activity inbrain

a) MMPs can cleave human APP₆₉₅ at the indicated position (arrow)between amino acid N504 and M505 in the N-terminal domain. The epitopefor the novel cleavage-specific antibody 10A8 is indicated (grey).sAPP-η of 80 kDa was specifically detected in DEA extracts of P10 WTmouse brain using the antibody 10A8, but was absent in APP KO brains.b-c) Soluble Aη levels, detected by the 9478D and M3.2 antibodies (Aη-α)were reduced in MT5-MMP −/− mouse brains, but unchanged in MT1-MMP −/−brains. Soluble (DEA) brain lysates were prepared from P10 mice. Totallevels of secreted APP (22C11), sAPP-α or sAPP-β were unchanged.

FIG. 3 shows that an inhibition of BACE1 results in elevated levels ofCTF-η and of Aη-α

a) Supernatants of CHO cells expressing human APP_(V717F) without orwith BACE1 inhibition (BI; 2 μM Merck IV) were compared to syntheticpeptides of Aη-β and Aη-α. Increased Aη-α peptide levels were observedupon BACE1 inhibitor treatment with 2D8 and 2E9 antibodies. The fragmentwith the lowest molecular weight, co-migrating with the syntheticpeptide Aη-β, disappeared upon BACE1 inhibition b) After overnightincubation of DIV 16 primary hippocampal neurons without or with theBACE1 inhibitor (BI; 2 μM Merck IV), supernatants were analyzed byWestern blotting. A strong increase of endogenous Aη-α was detected withM3.2 antibody. Total levels of secreted APP (22C11) were unchanged whilesAPP-α levels increased. The absence of sAPP-β and Aβ proves theeffective blockade of BACE1. c) Western blot analysis of DEA extracts ofP10 BACE1−/− mouse brains revealed a significant increase in Aη-αpeptides as compared to controls. Total levels of secreted APP (22C11)were unchanged while sAPP-α levels increased due to compensation byα-secretase activity. CTF-η levels were increased in RIPA lysates of theBACE1 KO mouse brain. As expected after an efficient BACE1 block, CTF-βand sAPP-β were severely reduced. d) BACE1 inhibition in vivo resultedin enhanced production of Aη-α species. A schematic presentation of thestudy design with a single oral dose treatment of APP_(V7171) mice withthe BACE1 inhibitor RO5508887 is shown in the upper panel. Inhibitortreated mice and vehicle treated controls were sacrificed and analyzedafter 5, 8 or 24 h. BACE1 inhibition reduced sAPP-β and CTF-β andincreased levels of Aη-α at 5 and 8 h after treatment. 24 h after thetreatment these changes were normalized due to the clearance of theinhibitor (a background band obtained with Y188 is indicated byasterisk).

FIG. 4 shows that Aη-α impairs hippocampal LTP.

a) Aη-α and Aη-β peptides were expressed in CHO cells and collected inOPTIMEM medium. Western blot analysis revealed the larger Aη-α and thesmaller Aη-β peptides. Higher molecular weight bands are most likely dueto posttranslational modification by glycosylation. b-e) SEC fractionsenriched for Aη were diluted (1:15) in ACSF for the treatment ofhippocampal slices and LTP measurements. Aη-α, Aη-β and control SECfractions (obtained from CHO cells transfected with the empty vector)were perfused over mouse hippocampal slices for 15 min after obtaining astable baseline of a fEPSP at the CA3-CA1 synapse. At the end of these15 minutes, a high-frequency stimulation protocol was applied (HFS; 2×(100 Hz, 1 s) at 20 second inter-stimulus interval) to induce long-termpotentiation (LTP). b) CHO supernatant by itself did not alter LTP whencompared to ACSF control. c) SEC fractions from conditioned media of CHOcells expressing Aη-α significantly inhibited LTP. d) SEC fractions fromconditioned media of CHO cells expressing Aη-β did not significantlyinhibit LTP. e) Quantification of LTP magnitudes (as % of baseline)calculated 45-60 minutes post-HFS from graphs in b-d with statisticalanalysis (*p<0.05); error bars represent s.e.m. n=number of fields.

FIG. 5 shows a table of antibodies used in this study.

List of antibodies used with indicated specificity, antibody epitope (ifknown) and dilutions applied in Western blotting and immunostainingtechniques.

FIG. 6 shows that dystrophic neurites in AD brains are positive forAη-epitope antibodies.

Immunohistochemistry with 22C11 (a, b), 9478D (c, d) and 9476M (e, f) inthe human hippocampus (CA1-subiculum region) of a control case (a, c, e)and in the AD case (b, d, f). Immuno-positive signals were observed with22C11 (a), 9478D (c) and 9476M (e) in the somata and neuropils of normaland AD brain. In AD brains these antibodies decorate dystrophic neurites(b, d, f). Calibration bar=30 μm.

FIG. 7 shows that Aβ_(S26C) dimers impair hippocampal LTP.

a) Treatment with Aβ_(S26C) dimers (100 nM final; JPT PeptideTechnologies, Germany; diluted in 25 ml re-circulating ACSF) reduced LTPas compared to interleaved LTP recordings in 25 ml re-circulating ACSF.b) Illustrated is the average LTP magnitude (at 45-60 minutes post-LTPinduction) normalized to pre-LTP baseline values (100%) in untreatedconditions (**p<0.01).

FIG. 8 shows high levels of Aη-α upon overexpression of MT1-MMP andMT5-MMP in N2a cells.

a, Expression of MT1-MMP and MT5-MMP in stably transfected N2a cellswere analyzed in lysates with specific antibodies. No major differencewas observed in the levels of APP-FL (APP full length) and APP-CTFs,while 2 predominant bands were observed in the range of 20-30 kDa. Withantibodies Y188 and with M3.2 an accumulation of CTF-η was detected inboth cell lines. While the smaller CTF-71 species was enriched in N2aMT1-MMP cells, the higher CTF-η species was enriched in N2a MT5-MMPcells. In lysates of N2a MT1-MMP cells additionally a 16 kDa fragmentwas identified by both antibodies and as APP-CTF derived from a cleavagecloser to the β-secretase cleavage site. b, In supernatants of N2a cellsexpressing either MT1-MMP or MT5-MMP the levels of sAPP-α and sAPP-1were substantially reduced, while sAPP-η was strongly increased asdetected with the antibody 10A8. The antibody M3.2, directed against themurine N-terminal Aβ domain, allowed to specifically detect a longerAη-α species of 16 kDa in the supernatants of N2a MT5-MMP cells, while aslightly shorter Aη-α species of 14 kDa was observed in the supernatantsof N2a MT1-MMP cells. Here additionally a 7 kDa species was detectedwith the antibody M3.2. While this peptide is most likely derived fromthe 16 kDa CTF observed in the N2a MT1-MMP lysates, the Aη-α species ofhigher or lower molecular weight correspond to the species seen incontrol N2a supernatants and fit to the different sizes found for theCTF-Aη species in the respective cell lysates.

Furthermore, reference is made to the following sequences which aregiven as exemplary embodiments:

All sequences are Homo sapiens sequences. It should be noted that thesequences indicated in the present application are denoted such thattheir N-terminal end is at the left side, and their C-terminal end is atthe right side. Hence, as an example, the sequence MISEPRISYG has theN-terminal end at M and the C-terminal end at G.

SEQ ID NO: 1: MISEPRISYG Epitope on Aη-peptide(s) SEQ ID NO: 2: DALMPSLTEpitope on Aη-peptide(s) SEQ ID NO: 3: PWHSFGADSVPEpitope on Aη-peptide(s) SEQ ID NO: 4: SEVKM Epitope on Aη-peptide(s)SEQ ID NO: 5: DAEFRHDSGYEVHHQK Epitope on Aη-peptide(s) SEQ ID NO: 6:MISEPRISYGNDALMPSLTETKTVELLPVNGEFSLDDLQPWHSFGADSVPANTENEVEPVDARPAADRGLTTRPGSGLTNIKTEEISEVKMExample of an Aη-β-peptide (residues 505-596 of P05067-4) SEQ ID NO: 7:MISEPRISYGNDALMPSLTETKTTVELLPVNGEFSLDDLQPWHSFGADSVPANTENEVEPVDARPAADRGLTTRPGSGLTNIKTEEISEVKMDAEFRHDS GYEVHHQKExample of an Aη-α-peptide (residues 505-612 of P05067-4) SEQ ID NO: 8:MISEPRISYGNDALMPSLTETKTTVELLPVNGEFSLDDLQPWHSFGADSVPANTENEVEPVDARPAADRGLTTRPGSGLTNIKTEEISEVKMDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITLVMLKKKQYTSIHHGVVEVDAAVTPEERHLSKMQQNGYENPTYKFFEQMQNExample of an CTF-η-peptide (residues 505-695 of P05067-4) SEQ ID NO: 9:EVPTDGNAGLLAEPQIAMFCGRLNMHMNVQNGKWDSDPSGTKTCIDTKEGILQYCQEVYPELQITNVVEANQPVTIQNWCKRGRKQCKTHPHFVIPYRCLVGEFVSDALLVPDKCKFLHQERMDVCETHLHWHTVAKETCSEKSTNLHDYGMLLPCGIDKFRGVEFVCCPLAEESDNVDSADAEEDDSDVWWGGADTDYADGSEDKVVEVAEEEEVAEVEEEEADDDEDDEDGDEVEEEAEEPYEEATERTTSIATTTTTTTESVEEVVRVPTTAASTPDAVDKYLETPGDENEHAHFQKAKERLEAKHRERMSQVMREWEEAERQAKNLPKADKKAVIQHFQEKVESLEQEAANERQQLVETHMARVEAMLNDRRRLALENYITALQAVPPRPRHVFNMLKKYVRAEQKDRQHTLKHFEHVRMVDPKKAAQIRSQVMTHLRVIYERMNQSLSLLYNVPAVAEEIQDEVDELLQKEQNYSDDVLANExample of an sAPPη-peptide (residues 19-504 of P05067-4) SEQ ID NO: 10:MLPGLALLLLAAWTARALEVPTDGNAGLLAEPQIAMFCGRLNMHMNVQNGKWDSDPSGTKTCIDTKEGILQYCQEVYPELQITNVVEANQPVTIQNWCKRGRKQCKTHPHFVIPYRCLVGEFVSDALLVPDKCKFLHQERMDVCETHLHWHTVAKETCSEKSTNLHDYGMLLPCGIDKFRGVEFVCCPLAEESDNVDSADAEEDDSDVWWGGADTDYADGSEDKVVEVAEEEEVAEVEEEEADDDEDDEDGDEVEEEAEEPYEEATERTTSIATTTTTTTESVEEVVRVPTTAASTPDAVDKYLETPGDENEHAHFQKAKERLEAKHRERMSQVMREWEEAERQAKNLPKADKKAVIQHFQEKVESLEQEAANERQQLVETHMARVEAMLNDRRRLALENYITALQAVPPRPRHVFNMLKKYVRAEQKDRQHTLKHFEHVRMVDPKKAAQIRSQVMTHLRVIYERMNQSLSLLYNVPAVAEEIQDEVDELLQKEQNYSDDVLANMISEPRISYGNDALMPSLTETKTTVELLPVNGEFSLDDLQPWHSFGADSVPANTENEVEPVDARPAADRGLTTRPGSGLTNIKTEEISEVKMDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITLVMLKKKQYTSIHHGVVEVDAAVTPEERHLSKMQQNGYENPTYKFFEQMQNHuman isoform APP695 of amyloid beta A4 protein(P05067-4)(residues 1-695)

The term “antibody,” as used herein, refers to an immunoglobulinmolecule which is able to specifically bind to a specific epitope on anantigen. Antibodies can be intact immunoglobulins derived from naturalsources or from recombinant sources and can be immunoreactive portionsof intact immunoglobulins. Antibodies are typically tetramers ofimmunoglobulin molecules. The antibodies used in the context of thepresent invention may exist in a variety of forms including, forexample, polyclonal antibodies, monoclonal antibodies, Fv, Fab andF(ab)₂, as well as single chain antibodies and humanized antibodies(Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: ALaboratory Manual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc.Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science242:423-426).

Antibodies may be generated using recombinant DNA technology, such as,for example, an antibody expressed by a bacteriophage as describedherein. The term “antibody” should also be construed to mean an antibodywhich has been generated by the synthesis of a DNA molecule encoding theantibody and which DNA molecule expresses an antibody peptide, or anamino acid sequence specifying the antibody, wherein the DNA or aminoacid sequence has been obtained using synthetic DNA or amino acidsequence technology which is available and well known in the art.

Monoclonal antibodies directed against full length or peptide fragmentsof a peptide or peptide may be prepared using any well known monoclonalantibody preparation procedures, such as those described, for example,in Harlow et al. (1988, In: Antibodies, A Laboratory Manual, Cold SpringHarbor, N.Y.) and in Tuszynski et al. (1988, Blood, 72:109-115).Quantities of the desired peptide may also be synthesized using chemicalsynthesis technology. Alternatively, DNA encoding the desired peptidemay be cloned and expressed from an appropriate promoter sequence incells suitable for the generation of large quantities of peptide.Monoclonal antibodies directed against the peptide are generated frommice or other animals, such as rabbits, hamsters, etc. immunized withthe peptide using standard procedures as referenced herein.

Nucleic acid encoding the monoclonal antibody obtained using theprocedures described herein may be cloned and sequenced using technologywhich is available in the art, and is described, for example, in Wrightet al. (1992, Critical Rev. in Immunol. 12(3,4):125-168) and thereferences cited therein. Further, the antibody of the invention may be“humanized” using the technology described in Wright et al., (supra) andin the references cited therein, and in Gu et al. (1997, Thrombosis andHematocyst 77(4):755-759).

To generate a phage antibody library, a cDNA library is first obtainedfrom mRNA which is isolated from cells, e.g., the hybridoma, whichexpress the desired peptide to be expressed on the phage surface, e.g.,the desired antibody. cDNA copies of the mRNA are produced using reversetranscriptase. cDNA which specifies immunoglobulin fragments areobtained by PCR and the resulting DNA is cloned into a suitablebacteriophage vector to generate a bacteriophage DNA library comprisingDNA specifying immunoglobulin genes. The procedures for making abacteriophage library comprising heterologous DNA are well known in theart and are described, for example, in Sambrook and Russell (2001,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y.).

Bacteriophage which encode the desired antibody, may be engineered suchthat the peptide is displayed on the surface thereof in such a mannerthat it is available for binding to its corresponding binding peptide,e.g., the antigen against which the antibody is directed. Thus, whenbacteriophage which express a specific antibody are incubated in thepresence of a cell which expresses the corresponding antigen, thebacteriophage will bind to the cell. Bacteriophage which do not expressthe antibody will not bind to the cell. Such panning techniques are wellknown in the art and are described for example, in Wright et al.,(supra).

Processes such as those described above, have been developed for theproduction of human antibodies using M13 bacteriophage display (Burtonet al., 1994, Adv. Immunol. 57:191-280). Essentially, a cDNA library isgenerated from mRNA obtained from a population of antibody-producingcells. The mRNA encodes rearranged immunoglobulin genes and thus, thecDNA encodes the same. Amplified cDNA is cloned into M13 expressionvectors creating a library of phage which express human antibodyfragments on their surface. Phage which display the antibody of interestare selected by antigen binding and are propagated in bacteria toproduce soluble human immunoglobulin. Thus, in contrast to conventionalmonoclonal antibody synthesis, this procedure immortalizes DNA encodinghuman immunoglobulin rather than cells which express humanimmunoglobulin.

Moreover, reference is made to the following examples which are given toillustrate, not to limit the present invention:

EXAMPLES Example 1

Material and Methods

Biochemical Methods

Soluble proteins were extracted from brain hemispheres with DEAbuffer³⁹, membrane proteins were extracted with RIPA buffer or byapplying a membrane preparation protocol as described⁴⁰. All Westernblot procedures were done essentially as described⁴¹.

BACE1 Inhibitor Treatment

Three-month-old heterozygous female transgenic mice FVB/N×C57Bl/6Jexpressing APP_(V7171) ²⁸ were used for BACE1 inhibition studies. Gavagemediated administration of BACE1 inhibitor RO5508887 (90 mg/kg, 14.06ml/kg) or vehicle (14.06 ml/kg) was performed once²⁷.

Slice Preparation and Electrophysiological Recordings

OPTIMEM was used to collect CHO cell supernatants. SEC was performedwith concentrated CHO supernatants using a FPLC (ÄKTApurifier) equippedwith a Superdex75 column (GE Healthcare). 1 ml samples were collectedwith a mobile phase flow rate set at 0.5 ml/min in standard ACSF. Acutehippocampal slices were prepared from Swiss mice (PND20-30) and kept inACSF⁴². Extracellular field excitatory post-synaptic potentials (fEPSPs)were obtained from CA1 pyramidal neurons. After 15 minutes of bathapplication of SEC fractions⁴³, LTP was induced by high frequencystimulation (2 pulses of 100 Hz for 1 second with a 20 secondinter-pulse interval). LTP was recorded for 60 minutes and statisticalanalysis was performed on the last 15 minutes of recording compared tobaseline fEPSP values.

Cell Culture

CHO and 7PA2⁴⁴ cells were grown in DMEM/F12 (Thermo Scientific)supplemented with 10% fetal calf serum (FCS, Thermo Scientific) plusPenicillin/Streptomycin and NEAA (Non-essential amino acids, PAA) in ahumid incubator with 5% CO₂ at a temperature of 37° C. For inhibitortreatment, cell culture medium was replaced with fresh, pre-warmed serumfree medium (OPTIMEM; Thermo Scientific) supplemented with inhibitors orDMSO as vehicle control. Treatment was initiated when cells reached90-100% confluency and conditioned media were harvested after 20-24 h.Supernatants were cleared by centrifugation (10′, 5500 g at 4° C.). Toobtain cell lysates, cell monolayers were washed once with ice-cold PBSand detached in 1 ml PBS using a cell scraper. The cell suspension waspelleted by centrifugation (5′, 1000 g at 4° C.) and lysed with RIPAbuffer (20 mM sodium citrate pH 6.4, 1 mM EDTA, 1% Triton X-100 inddH₂O) supplemented with Protease Inhibitor Cocktail (Sigma-Aldrich).The protein concentration of lysates was determined using the Uptima BCAssay Protein Quantitation kit (Interchim).

Primary Cell Culture

Hippocampal neurons were isolated from embryonic day 18 CD rats (CharlesRiver) as described⁴⁵. Dissociated neurons were plated at 17,700 cellsper cm² onto 6 cm dishes coated with poly-L-lysine (I mg/mL; Sigma) andcultured in Neurobasal medium supplemented with 2% B27 and 0.5 mML-glutamine (all from Invitrogen). Hippocampal cultures were maintainedin a humidified 5% CO₂ incubator at 37° C. For inhibitor treatment, DIV16 culture medium was replaced with fresh, pre-equilibrated N2 medium(supplemented with 20% of four days conditioned N2 medium from pureprimary cultured astrocytes) to which inhibitors or DMSO as vehiclecontrol were added⁴⁵.

Transgenic Mice, Animal Care, and Animal Handling

BACE1−/− and APPPS-21 mice were described before^(46,47) and were bredfor this study in a B16C57/J background. All treatments were approved bythe local committee for animal use and were performed in accordance tostate and federal regulations (license number KVR-1/221-TA116/09). Micehad access to pre-filtered sterile water and standard mouse chow (Ssnif®Ms-H, Ssniff Spezialdiaten GmbH, Soest, Germany) ad libitum and werehoused under a reversed day-night rhythm in IVC System Typ II L-cages(528 cm²) equipped with solid floors and a layer of bedding, inaccordance to local legislation on animal welfare.

BACE1 Inhibitor Treatment

APP_(V7171) ⁴⁸ mice were treated with the inhibitor RO5508887 providedby Hoffmann-La Roche⁴⁹. Three-month-old heterozygous female transgenicmice in mixed FVB/N×C57Bl/6J background expressing hAPP_(V7171) ⁴⁸ wereused for BACE1 inhibition studies. Gavage mediated administration ofBACE1 inhibitor (90 mg/kg, 14.06 ml/kg) or vehicle (14.06 ml/kg) wasperformed once⁴⁹. The BACE1 inhibitor was diluted in 5% ethanol (Merck)and 10% solutol (Sigma-Aldrich) in sterile water (Baxter). Animals weresacrificed after 5, 8 and 24 h. Mice were anesthetized with 3.5 μl pergram body weight of a mixture of ketamine (115 mg/ml ketaminehydrochloride, Eurovet), xylazin 2% (23.32 mg/ml xylazine hydrochloride,VMD Arendonk), atropine (0.50 mg/ml atropine sulphate, Sterop) andsaline (8:5:2:5, v/v/v/v). For brain preparation, mice were flushedtrans-cardially with ice-cold saline (3.5 ml/min, 3 min). The brain wasremoved from the cranium and dissected into left and righthemiforebrain, brainstem, cerebellum and olfactory bulb. The brainstructures were promptly immersed in liquid nitrogen and stored at −80°C. Different tissues (kidneys, spleen, liver, stomach, gut, lungs andheart) were examined and checked for gross abnormalities. No obviousabnormalities were observed in any of the treatment groups.

Preparation of Protein Extracts from Brain

Brains were removed from the cranium and dissected into left and righthemispheres.

Brain tissue was snap frozen in liquid nitrogen and stored at −80° C.Soluble proteins were extracted with DEA buffer (50 mM NaCl, 0.2%Diethylamine, pH 10+ protease inhibitor (P8340, Sigma-Aldrich)⁵⁰,membrane proteins were extracted with RIPA buffer (20 mM Tris-HCl (pH7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% NP-40.1% sodiumdeoxycholate, 2.5 mM sodium pyrophosphate+ protease inhibitor) orapplying a membrane preparation protocol as described before⁵¹.

Protein Analysis

Proteins were separated under denaturing conditions using discontinuousSDS-PAGE. Equal amounts of proteins denatured in Laemmli buffer wereloaded onto the gel and 10 μl of the SeeBlue Plus2 Prestained Standard(Invitrogen) served as molecular weight marker. Electrophoresis wasperformed in Tris-glycine buffer (25 mM Tris, 190 mM glycine in ddH₂O)using the Mini-PROTEAN system (BIORAD) on activated PVDF membranes. Lowmolecular weight proteins (<16 kDa) were separated using precastgradient Tricine Protein Gels (10-20%, 1 mm, Novex) in Tris-tricinebuffer using the XCell SureLock Mini-Cell system (Novex). Afterseparation by SDS-PAGE proteins were transferred onto membranes usingthe tank/wet Mini Trans-Blot cell system (BIORAD). CTFs, Aη and Aβ weredetected after transfer on Nitrocellulose membranes (Protran BA85; GEHlealthcare), while other proteins were blotted on PVDF (Immobilon-P,Merck Millipore). As size markers for Aη synthetic peptides Aη-β (92 aa;MISEPRISYGNDALMPSLTETKTTVELLPVNGEFSLDDLQPWHSFGADSVPANTENEVEPVDARPAADRGLTTRPGSGLTNIKTEEISEVKM⁹²) (=SEQ ID NO: 6) and the slightlylonger Aη-α (108 aa;MISEPRISYGNDALMPSLTETKTTVELLPVNGEFSLDDLQPWHSFGADSVPANTENEVEPVDARPAADRGLTTRPGSGLTNIKTEEISEVKMDAEFRHDSGYEVHIIQK¹⁰⁸) (=SEQ ID NO: 7)were obtained from Peptide Specialty Laboratories (PSL; Heidelberg,Germany). Upon completion of the transfer and prior to blocking,proteins transferred to nitrocellulose membranes were additionallydenatured by boiling the membrane in PBS (140 mM NaCl, 10 mM Na₂HPO₄,1.75 mM KH₂PO₄, 2.7 mM KCl in ddH2O, pH 7.4) for 5 min. After cooling toroom temperature nitrocellulose membranes as well as the PVDF membraneswere blocked in 1-Block solution (0.2% Tropix I-Block (AppliedBiosystems), 0.1% Tween20 in PBS) for 1 h at room temperature or o/n at4° C. (with agitation). Transferred proteins were detected usingimmunodetection and enhanced chemiluminescence (ECL). First, blockedmembranes were incubated with primary antibodies diluted in I-Blocksolution o/n at 4° C. (with agitation). After removal of the antibody,membranes were washed 3× in TBS-T buffer (10 min each, at roomtemperature, with agitation; 140 mM NaCl, 2.68 mM KCl, 24.76 mM Tris,0.3% Triton X-100 in ddH₂O, pH 7.6) and subsequently incubated with ahorseradish peroxidase (HRP) coupled secondary antibody (obtained fromPromega or Santa Cruz). Secondary antibodies were diluted in 1-Blocksolution and membranes were incubated for 1 h at RT (with agitation)followed by three washes in TBS-T. For ECL detection, membranes wereincubated with HRP substrate (ECL, GE Healthcare or ECL Plus, ThermoScientific) for 1 min at RT and signals were captured with X-ray films(Super RX Medical X-Ray, Fujifilm), which were subsequently developedusing an automated film developer (CAWOMAT 2000 IR, CAWO).

Human CSF Samples

Human CSF samples were collected at the Dept. of Neurology Outpatientunit for neurodegenerative disease (KBFZ) of the University of Bonn. CSFwas obtained by lumbar puncture at position L3, centrifuged and dividedin small aliquots. For further analysis, samples were stored at −80° C.Turbid or blood contaminated samples were excluded from analysis. Use ofthese samples for research purposes has been consented by all patientsaccording to the ethical committee requirements of the University ofBonn Ethical committee and approval number 279/10.

Neuropathology & Immunohistochemistry

Braak-NFT stages⁵², and CERAD⁵³ scores for neuritic plaques were used todetermine the degree of AD pathology according to the NIA-AAguidelines⁵⁴. Consecutive paraffin sections from the human medial lobewere stained with 22C11, 9476M and 9478D Primary antibodies weredetected with biotinylated anti-mouse and anti-rabbit IgG secondaryantibodies and visualized with avidin-biotin-complex (ABC-Kit, VectorLaboratories) and Diaminobenzidine-HCl (DAB). The sections werecounterstained with Haematoxylin. Positive and negative controls wereperformed. 9476M and 9478D stainings were assessed in 10 control and 10AD patient cases.

For double immunofluorescence analysis of APPPS-21 brain sections, 6month old mice were sacrificed by CO₂ inhalation according to animalhandling laws. Brains were dissected and fixed with 4% paraformaldehyde(PFA) in 0.1M PBS, pH 7.4 for 48 hours. For immunohistochemistry, 25μm-thick sagittal mouse brain cryosections were treated with 10 mMsodium citrate, pH 6 at 93° C. for 20 minutes, washed with 0.5% TritonX-100 in PBS, blocked with 5% goat serum (Invitrogen) and 0.5% TritonX-100 in PBS for 1 h and subsequently incubated overnight with primaryantibodies diluted in blocking solution. Primary antibodies were used aslisted in FIG. 5. DAPI was used to counterstain nuclei. Signals werevisualized using fluorescently labeled secondary antibodies. Confocalimages were acquired using a Plan-Apochromat 25×/0.8 oil differentialinterference contrast (DIC) objective on a LSM 710 confocal microscope(Zeiss) in sequential scanning mode using ZEN 2011 software package(black edition, Zeiss).

Laser Capture Microdissection (LCM)

For laser capture microdissection of plaque cores and halos, 14 monthsold transgenic APPPS-21 mice were used according to a previouslypublished protocol⁵⁵ with slight modifications. Mice brains weredissected, embedded in Shandon M1 embedding matrix (Thermo Scientific)and immediately frozen on crushed dry ice. 10 μm-thick sagittal sectionswere cut using a Microm HM 560 cryostat (Thermo Scientific), mounted onframe slides containing a 1.4 μm polyethylene terephthalate(PET)-membrane (Leica Microsystems) and subsequently stained or storedat −80° C. for later usage. Staining was performed as follows: brainsections were thawed shortly at room temperature, fixed with 75% ethanolfor 1 min, stained with 0.05% Thioflavin-S for 5 min, washed with 75%ethanol and dried at room temperature. LCM was performed on the same dayusing a laser dissection microscope (Leica, LMD 7000) with the followingsettings: excitation wavelength 495 nm, laser power 30, aperture 5,speed 6 and pulse frequency 119. Plaque cores and halos were cut using a63× magnification objective and control non-plaque areas using a 10×magnification objective and collected in 0.5 ml caps (Leica Microsystem)for protein analysis. Lysates were done essentially as described aboveusing RIPA with 0.1% SDS.

Slice Preparation and Electrophysiological Recordings

Transverse hippocampal slices (350 m) were prepared from postnatal day20-30 Swiss mice following standard procedures⁵⁶. Slices were cut inice-cold oxygenated (95% O2/5% CO₂) solution containing 206 mM sucrose,2.8 mM KCl, 1.25 mM NaH₂PO₄, 2 mM MgSO₄, 1 mM MgCl₂, 1 mM CaCl₂, 26 mMNaHCO₃, 0.4 mM sodium ascorbate, 10 mM glucose (pH 7.4). For recovery (1h), slices were incubated at 27° C. in oxygenated standard artificialcerebrospinal fluid (ACSF) containing: 124 mM NaCl, 2.8 mM KCl, 1.25 mMNaH₂PO₄, 2 mM MgSO₄, 3.6 mM CaCl₂, 26 mM NaHCO₃, 0.4 mM sodiumascorbate, 10 mM glucose (pH 7.4)⁵⁷. Slices were inspected in a chamberon an upright microscope (Slicescope, Scientifica Ltd) with IR-DICillumination and were perfused with the oxygenated ACSF at 27±1° C.Field excitatory post-synaptic potentials (fEPSPs) were recorded in thestratum radiatum of the CA1 region using a glass electrode (filled with1 M NaCl/10 mM HEPES, pH 7.4) and the stimuli (30% of maximal fEPSP)were delivered to the Schaeffer Collateral pathway by a monopolar glasselectrode (filled with ACSF). Electrodes were specifically placed justbelow the surface of the slice to maximize the exposure to circulatingpeptides. A minimum of 15 minutes stable baseline was first obtained instandard ACSF followed by another 15 minutes of bath application of ACSFcontaining SEC fractions (CHO, Aη-α or Aη-β; 1/15 dilution, interleavedrecordings) using re-circulation with a peristaltic pump at 2.5-3 mlmin⁻¹ while being continuously aerated with 95% oxygen. No alterationsin fEPSP baseline responses were observed after incubation with the SECfractions. In the continuous presence of the the ACSF/SEC solution, LTPwas then induced using a high frequency stimulation (HSF) protocol with2 pulses of 100 Hz for 1 sec with 20 see interval between pulses, andrecorded for one hour. Control recordings (no application of SECfractions) were obtained in an interleaved fashion where ACSF wasre-circulated using an identical procedure. For LTP analysis, the firstthird of the fEPSP slope was calculated in baseline condition (15minutes prior to induction of LTP and for 60 minutes post-induction).The average baseline value was normalized to 100% and all values of theexperiment were normalized to this baseline average (one minute bins).Experiments were pooled per condition and presented as mean±s.e.m. Dataanalysis was performed with the clampfit software (Molecular devices).Statistical analysis was performed using GraphPad (Prism 6) on the lastfifteen minutes of the recordings vs the 15 minutes of baseline using aone way-ANOVA and post-hoc Bonferroni test.

Example 2 Results

To identify novel proteolytic processing pathways of APP, the presentinventors searched for C-terminal fragments (CTFs) of APP different fromthose giving rise to p3 (CTF-α) or Aβ (CTF-β) in membrane fractions ofmouse brains¹⁵⁻¹⁷. Indeed, this revealed a novel CTF with an approximatemolecular weight of 30 kDa, recognized by an antibody to the C-terminusof APP (Y188), which is absent in the brains of APP knockout mice(APPKO)¹⁸ (FIG. 1b ; antibodies used are described in the table in FIG.5). The molecular weight of the novel CTF suggests a physiologicalcleavage of APP N-terminal to the known shedding sites of β-, andα-secretases, which the inventors named in analogy η-cleavage (FIG. 1a). In the soluble fraction, the present inventors detected theN-terminal cleavage product (sAPP-η; see FIG. 2a ), whose molecularweight of approximately 80 kDa clearly distinguishes it from alternativeN-terminal APP fragments described previously¹²⁻¹⁴. In addition, thepresent inventors observed lower molecular weight soluble fragments(Aη), which may derive from BACE1 (Aη-β) or ADAM10 (Aη-α) mediatedcleavage of CTF-η (FIG. 1a ). Aη was identified in the soluble fractionas several closely spaced peptides by antibody M3.2 (FIG. 1c ),demonstrating that some of these fragments contain the N-terminal partof the Aβ domain and are most likely ending at the α-secretase cleavagesite (see also FIGS. 1e and 3a ). Aη fragments were further validated byantibody 9478D directed against an epitope N-terminal to the Aβ domain(FIG. 1c ). The presence of endogenous CTF-η as well as Aη in brains ofmice suggests that Aη generation is a physiological processing pathwaysimilar to Aβ production^(19,20). To provide further evidence for Aηproduction in human brain, the present inventors analyzed cerebrospinalfluid (CSF). Robust signals of Aη were detected with the novel antibody2E9 (epitope was identified as indicated in FIG. 5) confirmingphysiological Aη production in vivo in humans (FIG. 1d ). In addition tothe physiological production of Aη in wild type mice and human CSF, thepresent inventors observed increased Aη synthesis in soluble fractionsof APPPS-21 transgenic mice²¹ as compared to wild type mice (FIG. 1e ).This allowed to identify the Aη-β species terminating at the BACE1cleavage site with the antibody 192swe²² (FIG. 1e ). Furthermore, ascompared to wild type mice, increased levels of CTF-η were observed inAPPPS-21 brain lysates (FIG. 1 f). Moreover, the present inventorsdetected co-staining of antibodies raised against the C-terminus of APP(Y188) with an epitope N-terminal to the Aβ domain (2E9) in dystrophicneurites of 6 month old APPPS-21 brains, but not in areas of aggregatedAβ detected by 6E10 staining in the plaque core (FIG. 1g ; similar datawere obtained with antibodies 22C11, 9476M, 9478D in human AD brains(FIG. 6)). This suggests that CTF-η may accumulate together withfull-length APP and CTF-α/β in dystrophic neurites surrounding neuriticplaques of APPPS-21 mice (FIG. 1g ). To provide direct evidence for theaccumulation of CTF-η in dystrophic neurites, the present inventors usedlaser capture microdissection (LCM) to differentially enrich forproteins accumulating in dystrophic neurites and plaque cores. Indeed,Western blot analysis revealed not only CTF-β and lower levels of CTF-α,but also CTF-η within the halo of dystrophic neurites and not within theplaque core area or regions devoid of plaques. As expected, Aβ wasobserved within the plaque core as well as in the surrounding halo (FIG.1h ).

Since membrane bound matrix-metalloproteinases like MT1-MMP and MT5-MMPwere shown to cleave in vitro at a site consistent with η-secretasecleavage^(23,24), the present inventors produced a neo-epitope specificantibody (10A8; FIG. 2a ), which allowed identification of theη-cleavage site. Antibody 10A8 identified a fragment corresponding tosAPP-η with an approximate molecular weight of 80 kDa in mouse brainlysates, which is absent in the APP KO brain (FIG. 2a ). Of note,antibody 10A8 did not identify sAPP-α/β (FIG. 2a ), demonstrating theselectivity of this antibody for the η-cleavage site. Thus η-secretasecleavage of APP occurs in vivo at least in part at amino acids 504/505(based on APP₆₉₅ numbering; the full APP₆₉₅ sequence is given in SEQ IDNO: 10). Moreover, sAPP-71 was found to be significantly increased insupernatants of cells co-expressing APP and MT1-MMP or MT5-MMP (data notshown). To prove which of the two MMPs initiates Aη production in vivo,the present inventors investigated brains from MT5- and MT1-MMP −/−mice^(25,26). Aη generation was reduced in brains from MT5-MMP knockoutanimals (FIG. 2b ), whereas a knockout of MT1-MMP had no significanteffect on Aη generation (FIG. 2c ). Thus MT5-MMP displays 7-secretaseactivity in brains, although additional η-secretases cannot be excluded.In fact, the results presented in FIG. 8 demonstrate that also MT1-MMPmay act as η-secretase, ultimately resulting in an Aη-α species of 14kDa that is slightly shorter than the Aη-α species that is obtained incells overexpressing MT5-MMP.

While investigating protease inhibitors capable to modulate η-secretaseactivity, the present inventors observed that pharmacological BACE1inhibition led to a pronounced accumulation of the long Aη-α species inCHO cells overexpressing APP_(V717F) (FIG. 3a ). This indicates that,upon BACE1 inhibition, processing by α-secretase leads to enhancedproduction of the long Aη-α species to the expense of shorter BACE1generated Aη-β. Similarly, in primary hippocampal neurons BACE1inhibition also led to an enhanced production of endogenous Aη-α (FIG.3b ). Moreover, analysis of brains from BACE1−/− mice confirmed a robustincrease of endogenous CTF-η and Aη-α in vivo (FIG. 3c ). Finally,pharmacological intervention in vivo with a single oral dose of theBACE1 inhibitor RO5508887²⁷ also revealed a significant time dependentincrease of CTF-η and Aη-α in APP7171 transgenic mice²⁸ (FIG. 3d ),which was fully reversible due to clearance of the inhibitor after 24 h.

Since cleavage products of the Aη processing pathway accumulate uponBACE1 inhibition (FIG. 3) and are observed in dystrophic neurites (seeFIGS. 1g and h ), the present inventors thought to investigate whethersoluble Aη peptides interfere with neuronal function similar to solubleAβ oligomers¹. LTP is a correlate of memory²⁹ and is frequently used toinvestigate neurotoxic effects of Aβ oligomers on learning exvivo^(5,30,31). In order to investigate potential effects of in vivoproduced A peptides on synaptic activity, the present inventorsexpressed Aη-β and Aη-α in CHO cells and collected conditioned media(FIG. 4a ) and enriched for these peptides by size exclusionchromatography (SEC). SEC fractions enriched for Aη-β and Aη-α wereapplied to hippocampal slices prior to inducing LTP in CA1 pyramidalneurons by high-frequency stimulation. The present inventors comparedLTP over 60 min obtained in the presence of Aη-β or Aη-α to LTP obtainedunder control conditions (FIG. 4b-e ). Strikingly, Aη-α inhibited. LTP(FIG. 4c ) to a degree comparable to Aβ_(S26C) ³¹ (FIG. 7), whereastruncated Aη-β had no effect (FIG. 4d ; quantified in FIG. 4e ).

The features of the present invention disclosed in the specification,the claims, and/or in the accompanying drawings may, both separately andin any combination thereof, be material for realizing the invention invarious forms thereof.

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1. A method of diagnosis and treatment, wherein said method comprises:detecting in cerebrospinal fluid (CSF) or plasma of a subject a) apeptide, or a collection of peptides, derived from amyloid precursorprotein (APP) by the action of matrix-metalloproteinase MT1-MMP orMT5-MMP, and by the action of at least one enzyme selected fromα-secretase (ADAM10) and β-secretase (β-site APP cleaving enzyme 1;BACE1); and/or b) a composition comprising one or more peptidesaccording to (a) and at least one further peptide derived from amyloidprecursor protein (APP) by the action of matrix-metalloproteinaseMT1-MMP or MT5-MMP only; and, if a) or b) are detected, treating thesubject by inhibiting the formation or action of said peptide or saidcollection of peptides or of said composition of peptides, and/orinhibiting the action or production of matrix-metalloproteinase MT5-MMPand/or MT1-MMP.
 2. The method according to claim 1, wherein said methodis used for the diagnosis and treatment of Alzheimer's disease.
 3. Themethod according to claim 1, wherein the peptide or collection ofpeptides or composition comprises a peptide selected from SEQ ID NOs: 1to
 9. 4. The method according to claim 3, wherein the peptide orcollection of peptides or composition comprises a peptide selected fromSEQ ID NO: 6 and SEQ ID NO: 7.